Antileishmanial Activity of Pyrazolopyridine Derivatives and Their Potential as an Adjunct Therapy with Miltefosine

Article abstract of DOI:10.1021/acs.jmedchem.6b01447

A series of pyrazolo(dihydro)pyridines was synthesized and evaluated for antileishmanial efficacy against experimental visceral leishmaniasis (VL). Among all compounds, 6d and 6j exhibited better activity than miltefosine against intracellular amastigotes. Compound 6j (50 mg/kg/day) was further studied against Leishmania donovani/BALB/c mice via the intraperitoneal route for 5 days and displayed >91 and >93% clearance of splenic and liver parasitic burden, respectively. Combination treatment of 6j with a subcurative dose of miltefosine (5 mg/kg) in BALB/c mice almost completely ameliorated the disease (>97% inhibition) by augmenting nitric oxide generation and shifting the immune response toward Th1. Furthermore, investigating the effect of 6j on Leishmania promastigotes revealed that it induced molecular events, such as a loss in mitochondrial membrane potential, externalization of phosphatidylserine, and DNA fragmentation, that ultimately resulted in the programmed cell death of the parasite. These results along with pharmacokinetic studies suggest that 6j could be a promising lead for treating VL as an adjunct therapy with miltefosine.

Full text of DOI:10.1021/acs.jmedchem.6b01447

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Article
Antileishmanial Activity of Pyrazolopyridine Derivatives
and their Potential as Adjunct Therapy with Miltefosine
Devireddy Anand, Pawan Kumar Yadav, Om P. S. Patel, Naveen Parmar, Rahul K. Maurya, Preeti
Vishwakarma, Kanumuri S. R. Raju, Isha Taneja, Muhammad Wahajuddin, Susanta Kar, and Prem P. Yadav
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01447 • Publication Date (Web): 06 Jan 2017
Downloaded from http://pubs.acs.org on January 7, 2017
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Antileishmanial Activity of Pyrazolopyridine Derivatives
and their Potential as Adjunct Therapy with Miltefosine
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†
,#
‡,#,┴
†
‡,┴
Devireddy Anand, Pawan Kumar Yadav,
Om P. S. Patel, Naveen Parmar, Rahul
†
‡,┴
§,┴
§,┴
K. Maurya, Preeti Vishwakarma,
Kanumuri S. R. Raju,
Isha Taneja,
M.
§
,┴
*,‡,┴
*,†,┴
Wahajuddin, Susanta Kar,
and Prem P. Yadav
†
Medicinal and Process Chemistry Division, CSIRꢀCentral Drug Research Institute, BSꢀ10/1,
Sector 10, Jankipuram Extension, Sitapur Road, Lucknow 226031, India.
‡
Parasitology Division, CSIRꢀCentral Drug Research Institute, BSꢀ10/1, Sector 10, Jankipuram
Extension, Sitapur Road, Lucknow 226031, India.
§
Pharmacokinetics and Metabolism Division, CSIRꢀCentral Drug Research Institute, BSꢀ10/1,
Sector 10, Jankipuram Extension, Sitapur Road, Lucknow 226031, India.
┴
Academy of Scientific and Innovative Research, Anusandhan Bhawan, New Delhi 110025,
India
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ABSTRACT. A series of pyrazolo(dihydro)pyridines were synthesized and evaluated for
antileishmanial efficacy against experimental visceral leishmaniasis. Among all, compounds 6d
and 6j exhibited better activity than miltefosine against the intracellular amastigotes. Compound
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6j (50 mg/kg/day) was further studied against Leishmania donovani/BALB/c mice via the
intraperitoneal route for 5 days and displayed >91 and >93% clearances in splenic and liver
parasitic burden, respectively. Combination treatment of 6j with the subꢀcurative dose of
miltefosine (5 mg/kg) in BALB/c mice almost completely ameliorated the disease (>97%
inhibition) by augmenting nitric oxide generation and shifting the immune response towards Th1
mode. Furthermore, investigating the effect of 6j on Leishmania promastigotes revealed that it
induced molecular events like a loss in mitochondrial membrane potential, externalization of
phosphatidylserine and DNA fragmentation that ultimately resulted in programmed cell death of
the parasite. These results along with pharmacokinetic studies suggested that 6j could be a
promising lead for treating VL as an adjunct therapy with miltefosine.
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INTRODUCTION
Leishmaniasis, designated as a neglected tropical disease (NTD) is a chronic infection caused by
an obligate intraꢀmacrophage protozoan parasite that belongs to the genus Leishmania, and in
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recent times has developed into a major health problem worldwide. Among all forms of
leishmaniasis, visceral leishmaniasis (VL) or kalaꢀazar is the most serious form of the disease. It
affects mainly the internal organs like liver, spleen and bone marrow resulting in a lethal
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condition if left untreated. An estimated 400,000 new cases of VL occurs each year all over the
world. Unfortunately, 90% of VL patients are present in just six countries, viz., Bangladesh,
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Brazil, Ethiopia, India, South Sudan and Sudan. It is characterized by irregular bouts of fever,
weight loss, enlargement of the liver and spleen, and anemia. The VL is mainly caused by L.
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donovani in the Indian subcontinent and East Africa. In the absence of effective and potential
vaccine candidates, chemotherapeutic regimens remain the mainstay of treatment for VL. In the
past, pentavalent antimonials (pentostam and glucantime) were recommended as the first line of
treatment for VL for more than 60 years, but the emergence of drugꢀresistant strains of the
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parasite have limited their use in endemic areas, especially in the Bihar state of India.
Therapeutic options for VL have been improved in recent years such as oral drug miltefosine, a
variety of liposomal formulations of amphotericin B, aminoglycoside antibiotic paromomycin,
and pentamidine. However, major concerns like teratogenicity, nephrotoxicity, hepatotoxicity,
long halfꢀlife, ototoxicity, parenteral administration for a longer duration, and unaffordable cost
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are associated with present antileishmanial chemotherapeutic agents. Therefore, there is an
urgent need of effective chemotherapeutics with improved pharmacological properties and
minimal side effects. Nowadays, combination therapy is one of the alternative approaches to
prevent the resistance developed by the parasite to monotherapy regimen, as it also increases the
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lifetime of existing drugs and shortens the duration of therapy. The parasite Leishmania has
established numerous mechanisms by which it inactivates the host protective immune response
to successfully survive within macrophages. A further outcome of the disease is regulated by the
induction of immunosuppressive molecules that includes transforming growth factor (TGF)ꢀβ,
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interleukin (IL)ꢀ10 released by Th2 cell population. These Th2 biasing molecules, in turn,
hamper the normal host immune response by inhibiting hostꢀprotective microbicidal molecules
like interferonꢀγ (IFN)ꢀγ, ILꢀ12, tumor necrosis factorꢀα (TNFꢀα), nitric oxide (NO) and reactive
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oxygen species (ROS). Unfortunately, chemotherapy against VL is observed to be associated
with various limitations. Thus the use of established antileishmanial agents at lower doses in
combination with novel agents that induced immunostimulation in adjunct with parasite killing
will be an effective strategy for the treatment of VL.
According to a recent review on the analysis of FDA approved drugs regarding structural
diversity and substitution pattern, the average numbers of nitrogen per drug is 2.5 for all the
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small molecule drugs. The literature reports on antileishmanial leads revealed that the nitrogen
containing heterocycles are very important class of compounds in antileishmanial drug
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discovery. Roy et al., reported that the diꢀindolylmethane (DIM) is a potent inhibitor of L.
donovani topoisomerase I enzyme, it binds strongly to the free enzyme and DIM stabilizes
topoisomerase IꢀDNA cleavage complexes in vitro and in vivo. Similarly,
dihydropyridopyrimidine class of compound exhibited in vitro antileishmanial activity in
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promastigote and amastigote models. Mello and coworkers studied the in vitro antileishmanial
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activity and QSAR properties of 4ꢀanilinopyrazolopyridineꢀ5ꢀcarboxylic esters. Kunick et al.,
demonstrated that 2ꢀ(3ꢀarylꢀ3ꢀoxopropenyl)ꢀ9ꢀtertꢀbutylꢀpaullones showed activity against both
axenic amastigotes and parasites in host macrophages without exhibiting toxicity to human host
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cells. Indoleꢀcontaining benzimidamide was identified as potential antileishmanial compound
possessing 10 times better activity and 401ꢀfold less toxicity than the drug pentamidine in cellꢀ
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based assays. Aminopyrazole ureas and quinazoline class of compounds have been identified
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as potential antileishmanial agents in a recent report. Based on the frequently encountered
antileishmanial scaffolds i.e. indole, pyrazole, and pyridine, we designed and synthesized indole
appended pyrazolodihydropyridines and pyrazolopyridines as antileishmanial prototypes (Figure
1).
N
N
HN
NH
N
O
Di-indolylmethane (ref. 10)
HN
O
Ar
H
N
Ar
O
N
N
N
HN
N
N
N
OH
O
Ar
Ar
O
R
4-Anilinopyrazolopyridine-5-
HN
R1
N
carboxylic ester (ref. 12)
N
H
H
prototype I
Prototype II
paullone (ref. 13)
Designed prototypes
S
NH
N
O
NH2
H
O
N
N
O
O
N
NH
O
N
N OH
N
N
N
N
H
N
F
O
R
Indole Scaffold Containing Antileishmanial
Benzimidamide (ref. 14)
Amino pyrazole urea (ref. 15)
Dihydropyridopyrimidine (ref. 11)
Figure 1. Designing of pyrazolodihydropyridine/pyrazolopyridine hybrids based on reported
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representative antileishmanial candidates.
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The designed prototypes can be accessed via multicomponent reaction (MCR) strategy using
suitable amine, aldehyde and 1,3ꢀdiketone source. MCRs have become a significant area of
research in organic and medicinal chemistry due to their atomꢀ and stepꢀeconomy and formation
of structurally diverse scaffolds for biological applications. This integrative nature of MCRs is
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attractive when a real need in molecular diversity exists. Herein, we report a MCR approach
towards the synthesis of indole appended pyrazolopyridines and pyrazolodihydropyridines and
their antileishmanial activity along with combination studies of most potent compound 6j with
miltefosine to achieve better therapeutic value.
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RESULTS AND DISCUSSION
Chemistry. Fused pyrazoles are an important scaffold in medicinal chemistry and tremendous
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efforts have been devoted in the literature to synthesize these molecules. In the present study,
pyrazolodihydropyridine and pyrazolopyridine compounds were synthesized as presented in
Scheme 1. The synthesis was initiated with the cyanoacetylation of indole 1 by using cyanoacetic
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acid in acetic anhydride at 60ꢀ70 °C by using reported procedure. The 3ꢀcyanoacetyl indole (2)
thus obtained was further treated with substituted aryl hydrazines in the presence of pꢀ
toluenesulfonic acid monohydrate (pꢀTsOH.H O) as the catalyst in EtOH under reflux conditions
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to afford the respective 1ꢀarylꢀ3ꢀindolylꢀ5ꢀamino pyrazoles 3 in excellent yields of >80%. The
reaction of 5ꢀaminopyrazoles 3, cyclic 1,3ꢀdiketones 4 and aryl aldehydes 5 in acetic acid at 100ꢀ
110 °C afforded the corresponding pyrazolopyridines 7 in good to excellent yields from 68% to
77% as mentioned in the experimental section. In few cases, the pyrazolodihydropyridine
derivatives 6 were formed as the major product even after prolonged reaction time. The
pyrazolodihydropyridines were further oxidized to aromatized compounds 7 in the presence of
2,3ꢀdichloroꢀ5,6ꢀdicyanobenzoquinone (DDQ) in acetonitrile. It was noteworthy to mention that
most of the compounds were purified via crystallization in EtOH, without the need of column
chromatography (see; experimental section). All the synthesized compounds were characterized
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by standard spectroscopic analysis such as H, C NMR, and ESIHRMS.
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a
Scheme 1. Synthesis of Pyrazolodihydropyridine and Pyrazolopyridine Derivatives
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Reagents and conditions. (i) cyanoacetic acid, Ac O, 60ꢀ70 °C, 10 min; (ii) substituted phenylhydrazines,
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pꢀTsOH, EtOH, reflux, 2 h; (iii) CH COOH, 100ꢀ110 °C, 2 h; (iv) DDQ, acetonitrile, RT, 2 h.
3
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Biological Activity. In Vitro Antileishmanial Activity of Pyrazolodihydropyridine and
Pyrazolopyridine Derivatives. The antileishmanial activity of pyrazolopyridine derivatives
(prototype I and II) was evaluated at 25 and 50 µM concentrations against extracellular
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promastigotes and intracellular amastigotes of luciferase expressing L. donovani. The
preliminary results revealed that nine compounds (6c, 6d, 6fꢀj, 7h and 7n) were found to be
active against promastigotes at the concentration of 50 µM with the inhibition rate between 80ꢀ
95% (Table 1). All the compounds were also examined for their efficacy against amastigote
stage of the parasite in L. donovani infected J774 macrophages. Among all, the compounds 6d
and 6j showed excellent activity with parasite killing >95% at 50 µM (Table 1). However, other
compounds 6c, 6fꢀi, 7h 7k and 7n exhibit less to moderate activity against intracellular
amastigotes stage with inhibition ranging from 28ꢀ60% at 50 µM (Table 1). Further, the half
maximal inhibition concentrations (IC ) of 6d and 6j were found to be 9.59 and 7.19 µM against
5
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promastigotes, and 7.36 and 4.05 µM against amastigotes, respectively, which is better than
antileishmanial drug miltefosine (antiamastigote IC : 9.46 µM) (Table 2). Furthermore, the MIC
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value was calculated by treatment of extracellular promastigotes and intracellular amastigotes
with six different concentrations of compounds 6d and 6j ranging from 200ꢀ6.25 µM by twoꢀfold
dilutions. We observed maximum parasite inhibition at a dose of 50 µM for the compounds 6d
and 6j, which was > 90 and > 95% for promastigotes and amastigotes, respectively. At higher
concentration (100 and 200 µM) the percentage inhibition of both promastigotes and amastigotes
by 6d and 6j did not alter significantly and was almost equal to 50 µM. Therefore, 50 µM doses
of both 6d and 6j were considered as MIC.
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Table 1.
In Vitro Antileishmanial Activity of Pyrazolodihydropyridines and
Pyrazolopyridine Derivatives
promastigotes
amastigotes
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growth inhibition (%)
growth inhibition (%)
entry
2
5 ꢁM
50 ꢁM
25 ꢁM
50 ꢁM
a
6a
62.5 ± 6.2
54.3 ± 5.4
79.3 ± 7.8
85.9 ± 5.9
52.3 ± 4.4
82.5 ± 5.8
85.5 ± 4.3
78.4 ± 6.5
79.1 ± 4.7
91.7 ± 5.2
56.5 ± 4.8
49.1 ± 5.6
54.2 ± 4.6
64.2 ± 6.5
65.3 ± 4.8
35.7 ± 6.7
44.6 ± 3.5
56.7 ± 4.8
61.3 ± 5.4
44.3 ± 3.9
50.7 ± 5.8
40.2 ± 4.7
84.1 ± 6.4
59.4 ± 5.8
59.1 ± 6.3
65.4 ± 5.3
65.3 ± 4.3
86.7 ± 2.8
90.6 ± 3.2
59.5 ± 4.3
86.5 ± 5.6
93.2 ± 5.1
81.2 ± 6.3
89.6 ± 3.2
93.3 ± 3.8
66.9 ± 4.9
62.4 ± 4.8
68.3 ± 4.7
67.4 ± 5.2
69.2 ± 3.9
40.6 ± 4.8
48.4 ± 5.3
67.4 ± 4.3
68.6 ± 3.6
52.0 ± 4.3
69.6 ± 4.9
54.3 ± 5.2
90.3 ± 3.8
74.1 ± 5.7
68.6 ± 6.1
NSI
NSI
NSI
28.2 ± 3.5
95.8 ± 2.5
NSI
6b
NSI
23.6 ± 2.9
91.5 ± 3.5
NSI
6c
6d
6e
6f
43.8 ± 3.8
37.3 ± 5.2
45.2 ± 4.3
25.2 ± 2.9
96.8 ± 2.2
NSI
51.7 ± 4.1
42.6 ± 4.5
59.6 ± 5.1
30.7 ± 3.8
97.3 ± 1.9
NSI
6g
6h
6i
6j
6k
6l
NSI
NSI
6m
NSI
NSI
6n
NSI
NSI
6
7
o
a
NSI
NSI
NSI
NSI
7b
NSI
NSI
7c
NSI
NSI
7d
NSI
NSI
7e
NSI
NSI
7f
NSI
NSI
7g
NSI
NSI
7h
22.4 ± 3.4
NSI
27.9 ± 2.8
NSI
7i
7j
NSI
NSI
7k
41.3 ± 4.9
61.2 ± 6.1
66.8 ± 5.2
52.4 ± 3.8
27.5 ± 4.6
44.6 ± 5.3
28.3 ± 6.7
49.8 ± 4.9
52.6 ± 5.3
100
51.8 ± 4.8
67.2 ± 5.3
72.3 ± 5.8
81.1 ± 4.7
33.4 ± 5.2
53.6 ± 4.8
41.1 ± 5.3
58.3 ± 5.1
63.4 ± 4.7
100
24.6 ± 3.3
NSI
41.3 ± 3.8
NSI
7l
7m
NSI
NSI
7n
20.8 ± 3.3
NSI
34.3 ± 3.7
NSI
7o
7
7
p
q
NSI
NSI
NSI
NSI
7r
7s
NSI
NSI
NSI
NSI
Miltefosine
99.8 ± 0.1
100
a
NSI (no significant inhibition). Percentage inhibitions are represented as means ± standard deviations for
at least three independent experiments for each compound.
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6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
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3
3
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3
3
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6
Table 2: Half Maximal Concentration (IC ) and Selectivity Index of Compounds 6d and 6j
5
0
cytotoxicity
selectivity index
SI=CC50/IC50
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(CC ꢁM)
50
antiamastigote
entry
a
IC (ꢁM)
5
0
J774
cells
482.5
409.4
55.7
Vero
cells
497.6
438.5
50.8
J774
cells
65.5
101
5.8
Vero
cells
67.6
108.2
5.3
6
6j
d
7.36
4.05
9.46
Miltefosine
a
IC50 for compounds 6d, 6j and miltefosine are represented as mean of three independent
experiments.
The antileishmanial activity follows a trend for the corresponding pyrazolodihydropyridine and
pyrazolopyridine compounds (Scheme 1).
In prototype I i.e. pyrazolodihydropyridines, by keeping a 1,3ꢀdiketone derived portion
(dimedone) constant, the compounds (6c, 6d and 6e) having electronꢀwithdrawing groups
(EWG) such as 4ꢀchloro, 3,4ꢀdichlorophenyl on R and electronꢀdonating groups (EDG) such as
1
2
,5ꢀdimethoxy, 3,4ꢀdimethoxy, 3,4,5ꢀtrimethoxyphenyl on R exhibited comparatively better
2
(52ꢀ90%) inhibition at 25 and 50 µM concentrations against promastigotes with respect to other
subprototypes. Compound 6d displayed excellent antileishmanial activity against amastigotes
(
IC = 7.36 µM; CC = 497 µM; SI = 67) as compared to the standard drug miltefosine (IC =
50 50 50
9
.46 µM; CC = 50 µM; SI = 5.3). Similar trends were observed in the case of compounds (6gꢀ
50
6k) derived from cyclopentane 1,3ꢀdione, having EWG (4ꢀfluoro, 4ꢀchloro, 3,4ꢀdichlorophenyl)
on the R and EDG (2,5ꢀdimethoxy, 3,4ꢀdimethoxy, 4ꢀhydroxyꢀ3,5ꢀdimethoxyphenyl) on R2
1
(Table 1 and 2). The compound 6j having 3,4ꢀdichlorophenyl on R and 3,4ꢀdimethoxy phenyl
1
on R showed excellent antileishmanial activity against amastigotes (IC = 4.05 µM; CC = 438
2
50
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µM; SI = 108) as compared to the standard drug miltefosine (IC = 9.46 µM; CC = 50 µM; SI
5
0
50
=
5.3).
In prototype II i.e. pyrazolopyridines, similar trends were observed as in the case of prototype I.
Compounds (7eꢀ7k) derived from dimedone possessing EWG (4ꢀfluoro, 4ꢀchloro, 3,4ꢀ
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dichlorophenyl) on R and EDG (4ꢀmethoxy, 2,3ꢀdimethoxy, 2,5ꢀdimethoxy, 3,4ꢀdimethoxy,
1
3
,4,5ꢀtrimethoxyphenyl) on R showed 41ꢀ90% inhibition against promastigotes. Compounds
2
(
7lꢀ7n) having EWG (4ꢀchloro, 3,4ꢀdichlorophenyl) on the R and EDG (2,5ꢀdimethoxy, 3,4ꢀ
1
dimethoxy, 3,4,5ꢀtrimethoxyphenyl) on R showed 52ꢀ81% inhibition against promastigotes.
2
Compounds (7oꢀ7s) having EWG (4ꢀfluoro, 4ꢀchlorophenyl) on R and EDG (4ꢀmethoxy, 2,5ꢀ
1
dimethoxy, 3,4ꢀdimethoxy, 2,3,4ꢀtrimethoxyphenyl) on R showed 27ꢀ63% inhibition against
2
promastigotes at 25 and 50 µM concentrations.
The effect of most active compounds (6d and 6j) on the inhibition of promastigote and
amastigote multiplication were also evaluated in a doseꢀ and timeꢀdependent manner by 3ꢀ(4,5ꢀ
dimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyltetrazolium bromide (MTT) assay and Giemsa staining,
respectively. We observed ˃95% inhibition against promastigotes (Figure 2A) and ˃93%
inhibition against intraꢀmacrophagic amastigotes (Figure 2B) in 72 h. Next, the safety index (SI)
of compounds 6d and 6j was investigated against both murine macrophages J774 and Vero cells.
The different cell types were treated with increasing concentrations of 6d and 6j and the
cytotoxicity was evaluated by MTT assay. Both compounds showed negligible toxicity against
J774 and Vero cells up to 200 µM concentrations as ˃95% cells are viable (see the supporting
information). The 50% inhibitory concentration (CC ) of 6d and 6j was found to be ˃400 µM
50
against both J774 and Vero cells (Table 2). The SI (CC /IC ) of 6d and 6j was found to be 65.5
50
50
and 88.4 in J774 cells, respectively, and 67.6 and 108.2 in Vero cells, respectively (Table 2). So,
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both these compounds were noncytotoxic and exhibited much higher safety index as compared to
the standard drug miltefosine (SI = 5.8 and 5.3 in J774 and Vero cells, respectively; Table 2).
Further to examine the effect of compounds on the growth pattern, promastigotes and
intracellular amastigotes in macrophages were treated with various concentrations of 6d and 6j,
ranging from 200 to 6.25 µM. It was observed that even at the lower concentrations (6.25 and
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12.5 µM), these compounds arrested the parasitic growth (Figure 2CꢀF). The results motivated us
to study the in vitro pharmacokinetics of compounds 6d and 6j prior to the in vivo studies in a
murine model of visceral leishmaniasis.
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Figure 2. In vitro antileishmanial efficacy and cytotoxicity assessment of 6j and 6d. (A) Percent viability
of promastigotes was evaluated by MTT method after treatment with IC and MIC of 6j and 6d for 12ꢀ72
5
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h. (B) J774 macrophages were infected with promastigotes (1:8) as described in methods followed by
treatment with IC50 and MIC doses of compounds 6j and 6d. The number of intraꢀmacrophagic
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amastigotes was determined by Giemsa staining. (C, D) Promastigotes were treated with various
concentrations of 6j (C) and 6d (D) and cell viability was determined by luciferase activity. (E, F)
Intracellular amastigotes were treated with same concentrations of 6j (E) and 6d (F) and parasitic growth
was estimated on the basis of relative luminescence units in lysed macrophage cells. Data are
representative of three independent experiments and results shown are means ± SD for each time point.
Significance shown are for untreated vs treated groups, ***p<0.0001.
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In Vitro Pharmacokinetic Study. To assess the stability of the compounds in different
conditions, 6d and 6j were studied in simulated gastric fluid (SGF, pHꢀ1.2) and intestinal fluid
(
SIF, pHꢀ6.5). The metabolic stability of the molecules was also assessed in mice S9 fraction to
see the contribution of liver in the elimination of the compounds. Upon incubation with SGF,
5% of 6j, and 75% of 6d were found remaining intact after 120 min, suggesting that both these
8
compounds would be stable in acidic pH that would be encountered in the stomach. Upon
incubation with SIF, 99% of 6j and 58% of 6d were found remaining intact after 120 min,
suggesting that compound 6d is less stable in basic pH that would be encountered in the
intestine. The representative plots of SGF and SIF stability are mentioned in Figure 3A and B.
Around 45 and 31% of the parent compounds 6j and 6d, respectively, were found intact after 60
minutes of incubation with mice S9 fraction. In vitro metabolic halfꢀlife and rate constant for
metabolism is given in Table 3 and the representative percent parent remaining vs time plots are
given in Figure 3C. Based on the preclinical stability studies compound 6j demonstrated better
stability in different conditions compared to 6d.
Table 3. In Vitro Metabolic Stability Parameters of 6j and 6d
parameter
6j
6d
ꢀ1
k (min )
0.013
0.017
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In vitro halfꢀlife, t1/2 (min)
Intrinsic clearance, CL (µL/min/mg protein)
50.45
27.47
39.99
34.65
int
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Figure 3. Percent parent remaining vs time plots of 6j and 6d upon incubation. (A) incubation in
simulated gastric fluid. (B) incubation in simulated intestinal fluid. (C) incubation with mice S9 fraction.
In Vivo Activity and Combination Therapy of Compound 6j against L. donovani/Mice
Model. As described in materials and methods (experimental Section), compounds were
administered intraperitoneally in mice infected with L. donovani with a dose range of 12.5ꢀ200
mg/kg/day for 5 consecutive days and sacrificed at day 7 after last treatment. The Parasitic
burden was studied in liver and spleen smears. There was a clear dose oriented inhibition in the
parasitic burden in the mice treated with 6d and 6j and both the compounds were seen to inhibit
the parasitic burden in both liver and spleen (Figure 4A). But both compounds displayed
different doseꢀresponse curves as 6j showed better antileishmanial efficacy compared to 6d.
Compound 6j showed >50% parasitic inhibition at a much lower dose of 25 mg/kg in both liver
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and spleen whereas for 6d at least 100 mg/kg was required to eliminate >50% parasite in both
liver and spleen. Moreover, at a dose of 50 mg/kg/day for 5 days, 6j showed >91 and >93%
clearance in splenic and liver parasite burden, respectively (Figure 4A). Further increasing the
dose of 6j could not significantly enhance its therapeutic efficacy (90 and 92.7% in spleen and
liver, respectively at 100 mg/kg and 90 and 93% in spleen and liver, respectively at 200 mg/kg)
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(Figure 4A). On the contrary, the maximum inhibition of liver and splenic parasite burden for 6d
was 62 and 68%, respectively at a much higher dose (200 mg/kg) (Figure 4A). As 6j showed
superior antileishmanial efficacy than 6d, we performed time kinetics of organ parasite burden
after 6j treatment at its optimal dose i.e. 50 mg/kg. The compound was observed to inhibit the
parasitic burden by >93% in both liver and spleen as early as 4 weeks postꢀinfection i.e. almost
oneꢀweek postꢀtreatment (Figure 4B and C). However, increasing the time points did not
significantly increase in the inhibition of parasitic burden in both liver and spleen as observed up
to 7 weeks of postꢀtreatment (Figure 4B and C).
Nowadays, in order to minimize the side effects caused by a drug, the combination therapy using
a lower dose of standard drugs is going popular which reduces the dose, time and side effects.
Despite its strong antileishmanial efficacy, standard oral drug miltefosine is demonstrated to
have a serious side effect in human i.e. teratogenicity. It was observed that compounds 6d and 6j
showed different doseꢀresponse curves whereas 6j showed better antileishmanial efficacy
compared to 6d. We, therefore, thought it was worthwhile to check whether a lower dose of
miltefosine in combination with 6j could ameliorate in vivo parasite burden in BALB/c mice,
similar to a higher curative dose of miltefosine. Percentage reduction in parasitic burden in the
group treated with 6j (25 mg/kg) was >59 and >48% in spleen and liver, respectively at day 7, so
this dose was considered as a subꢀcurative (Figure 4D and E). A dramatic inhibition was
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observed in parasitic burden in the infected groups treated with combination of subꢀcurative
miltefosine (5 mg/kg) and 6j (25 mg/kg) at both day 4 and day 7ꢀpostꢀtreatment in liver and
spleen (>92 and >97% inhibition, respectively as compared to infected mice) and the efficacy is
well comparable with the curative dose of miltefosine i.e. 25 mg/kg (>98% inhibition in both
liver and splenic parasite burden) (Figure 4D and E). Treatment with the subꢀcurative miltefosine
resulted in a moderate parasitic inhibition in both liver (>75%) and spleen (>61%) at day 7
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(Figure 4D and E). Throughout the course of the experiment at postꢀtreatment, the animals
seemed to be completely healthy with no variation in normal body weight. Both liver and spleen
weights of the treated groups were significantly reduced compared to infected control after mono
and combination therapy. All results showed better scope for combination therapy and motivated
us to evaluate the status of host protective immune responses against VL in different
experimental groups in detail.
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Figure 4. Effect of 6j and 6d monotherapy or combination therapy against on parasitic burden of L.
donovani infected BALB/c mice. L. donovani infected mice were treated with various combinations of 6j,
6d and miltefosine as described in materials and method. (A) 15 days infected BALB/c mice were treated
with different doses of 6j and 6d as indicated. Splenic and hepatic parasite loads were determined at day 7
postꢀtreatment by a stampꢀsmear method. (4BꢀC) At various time points after treatments, mice of different
experimental groups were sacrificed for determination of hepatic (B) and splenic (C) parasite loads by a
stampꢀsmear method and expressed as Leishman Donovan units (LDU). (DꢀE) L. donovani infected mice
were treated with various combinations of 6j and miltefosine and splenic (D) and hepatic (E) parasitic
burden was estimated as indicated. 1 LDU = amastigotes per nucleated cell × organ weight in milligrams.
Data represents mean ± SD (n = 5 mice per group), representative of three independent experiments.
Significance shown are for infected vs various treatment groups and subꢀcurative dose of miltefosine vs
combination group, *p < 0.05, **p < 0.005, ***p < 0.0001.
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Effect of Combination Therapy on T Cell Proliferation and Th1/Th2 Cytokine Balance In
Vivo. Disease progression in VL is associated with impaired T cell proliferation and weak cellꢀ
mediated immunity, while disease healing demands higher T cell proliferation to exhibit the
appropriate immune response. Therefore, the effect of combination therapy was investigated in
the restoration of T cell proliferation in murine EVL. As observed, splenocytes isolated from L.
donovani infected mice were unable to initiate T cell proliferation in the presence of SLA
(soluble L. donovani promastigote antigen). Whereas the groups treated with both subꢀcurative
miltefosine (5 mg/kg) and 6j (25 mg/kg) exhibited 9.9ꢀ and 8.4ꢀfold increase in T cell
proliferation compared to the infected group when stimulated by SLA at day 4 and day 7,
respectively (Figure 5A). This was even higher than curative miltefosine (25 mg/kg) which
resulted in 8.2ꢀ and 7.1ꢀfold increase at day 4 and day 7, respectively. T cell proliferation was
moderate (2.2ꢀ to 3.4ꢀfold) in infected mice treated with lower doses of 6j or subꢀcurative
miltefosine cell proliferation (Figure 5A). L. donovani switches the Th1/Th2 cytokine balance
towards antiꢀinflammatory Th2 mode to survive and propagate successfully inside its host.
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Successful inhibition of the parasite demands higher levels of Th1 cytokines. A detailed splenic
cytokine study was done to further assess the kind of immunological response in splenocytes of
the mice with various treatment regimens. The cytokine profiling by ELISA revealed that the
treatment with combination of subꢀcurative miltefosine and 6j resulted in 9.1ꢀ, 7.4ꢀ and 12.7ꢀfold
increase in the level of IFNꢀγ, TNFꢀα and ILꢀ12, respectively, at day 4 postꢀtreatment which was
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.5ꢀ, 7.3ꢀ and 11.3ꢀfold increase at day 7 postꢀtreatment as compared to infected control (Figure
B, C, and D). However, the same combination therapy resulted in 88 and 91% decreased at day
5
4 as well as 93ꢀ and 94%ꢀfold decreased at day 7 in the level of TGFꢀβ and ILꢀ10, respectively
Figure 5E and F). These data were similar to cytokine secreted by infected mice treated with
(
curative miltefosine (25 mg/kg) which resulted in 9.1ꢀ, 6.5ꢀ and 12.9ꢀfold increase in the level of
IFNꢀγ, TNFꢀα and ILꢀ12 at day 4 while 8.1ꢀ, 5.3ꢀ and 9.5ꢀfold increaseat day 7, respectively
(
Figure 5B, C and D). Similarly, curative miltefosine decreased the level of Th2 cytokine in
infected mice likewise combination therapy group (87.5 and 88.7% decreases at day 4 as well as
4.6 and 92.9% decreases at day 7 in the level of TGFꢀβ and ILꢀ10, respectively) (Figure 5E and
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F). Subꢀcurative doses of miltefosine as well as 6j showed a similar trend for Th1/Th2 cytokine
synthesis but to lower extent which was consistent with their moderate inhibition of splenic and
liver parasite burden. Collectively, these results suggest that in the in vivo situations, the
combination of 6j with subꢀcurative miltefosine restores Tꢀcell proliferation towards host
protective Th1 type thereby successfully eliminates the parasite from the infected mice.
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Figure 5. Effect of 6j/miltefosine combination therapy on Tꢀcell proliferation and Th1/Th2 cytokine
balance in L. donovani infected BALB/c mice. Infected BALB/c mice were treated with different
combinations of 6j and miltefosine and splenocytes were isolated for assays. Isolated splenocytes from
different groups on day 4 and day 7 post treatments were cultured and stimulated with 5 µg/ml soluble
leishmanial antigen (SLA) for 72 h at 37 °C/5% CO incubator. (A) T cell proliferation was assessed by
2
MTT assay. (BꢀF) Supernatants from cultured splenocytes were evaluated for determination of IFNꢀγ (B),
TNFꢀα (C), ILꢀ12 (D), TGFꢀβ (E) and ILꢀ10 (F) levels by sandwich ELISA. Data represents mean ± SD
(n = 5 mice per group), representative of three independent experiments. Significance shown are for
infected vs various treatment groups and subꢀcurative dose of miltefosine vs combination group, *p <
0.05, **p < 0.005, ***p < 0.0001, ns = nonꢀsignificant.
iNOS Expression and NO Generation Following Combination Therapy. iNOS mediated NO
generation is crucial for controlling Leishmania infection in a murine model of EVL. Progression
or healing of the disease depends on the nitrosative stress mediated by macrophageꢀderived NO
critical for killing of intracellular L. donovani trapped in phagolysosomes. Therefore, the effect
of combination therapy was explored on iNOS gene expression and NO generation. iNOS gene
transcription in the murine splenocytes isolated from different treated groups coincided with the
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level of pathogen clearance. The maximum increase in mRNA level was observed in the group
treated with subꢀcurative doses of miltefosine plus 6j (11.8ꢀ and 8.5ꢀ fold at day 4 and day 7,
respectively) and curative miltefosine (12.3ꢀ and 9.1ꢀfold at day 4 and day 7, respectively)
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(
Figure 6A). This was fairly higher from the level of iNOS mRNA expression in infected animal
treated with subꢀcurative miltefosine (6.4ꢀ and 3.4ꢀfold at day 4 and day 7, respectively) and 25
mg/kg of 6j (4.9ꢀ and 3.3ꢀfold at day 4 and day 7, respectively) (Figure 6A). Similarly, NO
generation in the culture supernatant of mice splenocytes from various therapeutic groups
reflected the level of protection. Leishmania infection failed to induce nitrite production
following SLA stimulation as expected while this was counteracted in the treated groups. The
maximum generation of nitric oxide was seen in the mice treated with curative miltefosine as
well as the mice treated with a combination of both subꢀcurative miltefosine and 6j (25 mg/kg) at
day 4 (7.4ꢀ and 5.6ꢀfold increase, respectively compared to infected untreated) in SLAꢀ
stimulated splenocytes (Figure 6B). Similarly, the mice treated with 25 and 50 mg/kg of 6j and
subꢀcurative miltefosine showed significantly more nitric oxide compared to the infected control
(4.1ꢀ, 4.7ꢀ and 4.3ꢀfold increase, respectively (Figure 6B). These observations confirmed the
heightened host immuneꢀaccessory functions of the infected animals undergoing combination
therapy.
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Figure 6. Effect of 6j/miltefosine combination therapy on iNOS expression and NO generation in L.
donovani infected BALB/c mice. (A) RNA was isolated from control, infected and treated BALB/c mice
splenocytes on day 4 and day 7 post treatments and mRNA expression of iNOS was evaluated by realꢀ
time PCR. The mRNA level was normalized to mouse βꢀActin and expressed as a fold change compared
with uninfected controls. (B) Griess assay was performed to measure nitric oxide level in splenocytes
supernatants from different groups of mice stimulated with SLA (5 µg/ml) for 72 h as described in
methods. Data represents mean ± SD (n = 5 mice per group), representative of three independent
experiments. Significance shown are for infected vs various treatment groups and subꢀcurative dose of
miltefosine vs combination group, *p < 0.05, **p < 0.005, ***p < 0.0001, ns = nonꢀsignificant.
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Induction of Apoptotic Cascades in Leishmania donovani Promastigotes Following
Treatment with 6j. It is well established that cyclic pyrazole compounds induce apoptosis
20
cascades in various cancer cells and effective parasite suppression by compound 6j in both in
vitro and in vivo model of VL prompted us to investigate the programmed cell death mechanisms
in L. donovani promastigotes. The flipping of phosphatidylserine from the inner leaflet to the
outer leaflet of the plasma membrane is an early event of apoptosis. Using FITC conjugated
annexin V which binds to phosphatidylserine and PI which binds to DNA in flow cytometry,
ꢀ
ꢀ
three subpopulations can be distinguished, viable promastigotes (annexin V /PI ), early apoptotic
+
ꢀ
+
+
promastigotes (annexin V /PI ) and late apoptotic or necrotic promastigotes (annexin V /PI ).
The Figure 7A clearly demonstrated that 38.2% of L. donovani promastigotes undergo apoptosis
when treated for 48 h with IC50 dose of compound 6j where 21% cells undergo late apoptosis
and 17.2% cells are in early apoptotic phase. Furthermore, treatment with maximum inhibitory
concentration (50 µM) of 6j for 48 h induced 65.8% apoptosis in promastigotes where 11.6 and
54.2% cells are in early and late apoptotic cascade, respectively. On the contrary, only 8.2% of
the parasite treated with vehicle (0.1% DMSO) undergoes apoptosis. DNA fragmentation is a
hallmark feature of apoptosis in metazoan as well as unicellular protozoans, thus to further
reconfirm apoptotic induction mediated by compound 6j in the nuclear material of L. donovani
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promastigotes, TUNEL assay was performed to detect the free ends of cleaved DNA fragments.
As shown in Figure 7B, promastigotes incubated with both IC50 and 50 ꢁM of compound 6j
showed a large number of bright fluorescent nuclei (indicating fragmentation of DNA) and
significantly higher percentage of TUNEL positive cells as compared to the untreated and
vehicleꢀtreated promastigotes where fewer labeled nuclei were observed. In addition, bright field
microscopic examination also showed that treated promastigotes were round in shape as
compared to elongated and flagellated nonꢀtreated promastigotes.
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Nevertheless, dysfunction of mitochondrial events has been shown to participate in the induction
of apoptotic process. One of the early events responsible for induction of apoptosis cascade is a
21
loss of MMP (ꢁΨ ) and it plays an important role in drugꢀinduced death also in Leishmania.
m
To investigate whether compound 6j treatment could disrupt MMP in L. donovani promastigotes,
a time course study of ꢁΨ measurement was performed using the fluorescent dye, JCꢀ1. JCꢀ1 is
m
a lipophilic cationic dye that fluoresces red upon aggregation in healthy cells, and however, after
a loss of ꢁΨ , it forms monomer throughout the cell and emits green fluorescence. We observed
m
that incubation of promastigotes with compound 6j at 50 ꢁM for different time periods resulted
in significant drop in transmembrane potential. Upon flow cytometric analysis it was observed
that 6j treatment to L. donovani promastigotes remarkably shift the fluorescence emission from
red to green where 15.2% cells showed green fluorescence at 6 h postꢀtreatment whereas 32.9
and 52.6% cells showed green fluorescence at 12 and 24 h, respectively (Figure 7C). On the
contrary 5.02% of the parasite treated with vehicle (0.1% DMSO) showed green fluorescence at
24 h. Further incubation for 48 h lead to a little decrease in green fluorescence (44.9%) (Figure
7
C). Taken together, we can conclude that compound 6j induce disruption of ꢁΨ as an early
m
event to trigger apoptosis of Leishmania parasite.
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Figure 7. Compound 6j induced apoptotic cell death cascade in L. donovani promastigotes. (A)
Promastigotes were incubated with IC and MIC dose of compound 6j (50 ꢁM) and DMSO (0.1%) for 48
50
h coꢀstained with annexin Vꢀ FITC and propidium iodide. Stained promastigotes were analyzed using
FACS Calibur Flow Cytometer and data was analyzed with Cell Quest software. (B) Exponential phase
promastigotes were treated with compound 6j (IC50 and MIC) for 48 h. Control promastigotes were
treated with 0.1% DMSO containing M199 medium. Cells were observed under both phase contrast and
fluorescent microscopy. Green color indicates TUNELꢀpositive L. donovani promastigotes (C)
Exponential phase Dd8 promastigotes were treated with compound 6j (50 ꢁM) for 6ꢀ48 h and were
labeled with potentiometric probe JCꢀ1(2 ꢁM final concentration).Timeꢀdependent alterations in relative
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(ꢁΨ ) are expressed as the ratio of fluorescence measurements at 590 vs 530 nm. All results are
m
representative of three independent experiments.
In Vivo Pharmacokinetic Study. The mean plasma concentrationꢀtime profile of 6j upon oral
administration at a dose of 50 mg/kg is as shown in Figure 8 and its estimated pharmacokinetic
parameters are represented in Table 4. Results show its faster absorption (Tmax, 1.75 ± 0.50 h)
with a peak plasma concentration level of 4.88 ± 0.61 µM (Cmax). The time taken for the
systemic levels to reduce to half (halfꢀlife, t ) was 4.81 ± 0.29 h and the elimination rate
1
/2
ꢀ1
constant (K ) was found to be 0.14 ± 0.01 h . The apparent volume of distribution was found to
e
be 19.56 ± 2.36 L/kg and clearance was found to be 2.84 ± 0.48 L/h/kg indicating that the
compound showed low clearance. The area under the concentrationꢀtime profile (AUC0ꢀ∞
)
representing the total systemic drug exposure was 31.67 ± 6.22 h*µM. The pharmacokinetic
parameters indicate that the compound 6j have better oral exposure and can be a better lead for
further optimization.
Table 4. Pharmacokinetic Parameters of 6j upon Oral Administration at 50 mg/kg Dose.
Data Represented as Mean ± S.D. (n=4)
Parameter
Estimate
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K (1/h)
0.14 ± 0.01
4.81 ± 0.29
1.75 ± 0.5
e
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Tmax (h)
Cmax (µM)
4.88 ± 0.61
31.67 ± 6.22
19.56 ± 2.34
2.84 ± 0.48
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j Oral Profile at 50 mg/kg
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Time (h)
Figure 8. Mean plasma concentrationꢀ time profile of 6j upon oral administration. Data represented as
mean ± S.D. (n=4).
CONCLUSION
A series of pyrazolodihydropyridines and pyrazolopyridines were synthesized and tested for their
in vitro antileishmanial activity against luciferase expressing L. donovani. Compounds 6d and 6j
were found to be most promising as compared to the drug miltefosine. Compounds 6d and 6j
exhibited much high SI value and good safety profile than miltefosine. The in vivo study of
compounds 6d and 6j was performed in L. donovani/mice model via ip route, in which
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compound 6j showed better activity up to day 28 postꢀtreatment as compared to the 6d. The
combination studies of the most active compound 6j (25 mg/kg) and a subꢀcurative dose of
miltefosine (5 mg/kg) exhibited comparable effects to that of a curative dose of standard
marketed drug miltefosine (25 mg/kg) against L. donovani infected mice. The molecular and
immunological studies showed that compound 6j has a dual nature to act as a direct parasite
killing agent as well as host immunostimulant. Pharmacokinetic parameters and in vivo studies of
compound 6j suggested that it may be a potential lead for further optimization to identify
candidates for the treatment of the visceral leishmaniasis.
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EXPERIMENTAL SECTION
General Methods. All reagents were purchased from commercial sources and used without
further purification. Reactions were monitored by thinꢀlayer chromatography (TLC) silica gel
plates with fluorescence F . Melting points were taken in open capillaries on melting point
254
apparatus and are presented uncorrected. Infrared spectra were recorded using FTꢀIR
1
spectrophotometer. H NMR spectra were recorded on a 300, 400 and 500 MHz Bruker Avance
1
3
spectrometer and were determined in CDCl and DMSOꢀd . C NMR spectra were recorded in
3
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DMSOꢀd and CDCl on a 50, 75, 100, and 125 MHz Bruker Avance spectrometer. Twoꢀ
6
3
dimensional (2D) experiment HSQC was performed on a 100 MHz Bruker Avance spectrometer.
Chemical shifts are reported in parts per million. Splitting patterns are described as singlet (s),
broad singlet (br s), doublet (d), broad doublet (br d), double doublet (dd), triplet (t), quartet (q),
and multiplet (m). HRESIMS spectra were recorded using QꢀTOF mass spectrometer. All tested
compounds were ≥95% pure by HPLC with detection at 254 nm and 220 nm. The target
compounds and substructures presented herein are not associated with commonly encountered
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PAINS . The potent compounds 6d and 6j passed the PAINS filter using SYBYLꢀXꢀ2.1
SYBYLꢀX Suite. available online: http://tripos.com).
General Procedure A. Synthesis of 1ꢀarylꢀ3ꢀindolylꢀ5ꢀamino pyrazoles (3aꢀc). To a solution of
ꢀ(cyanoacetyl)indole 2 (1.0 equiv.) in EtOH (20 mL) was added substituted phenylhydrazine
hydrochloride (1.1 equiv) and pꢀTsOH.H O (0.3 equiv.). The reaction mixture was refluxed for
(
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h. After completion of the reaction (monitored by TLC), the reaction mixture was cooled to
room temperature and concentrated under reduced pressure. The residue was neutralized with a
saturated NaHCO solution (3 x 20 mL), extracted with EtOAc (3 x 20 mL), dried over
3
anhydrous Na SO , and concentrated under reduced pressure. The pure 1ꢀarylꢀ3ꢀindolylꢀ5ꢀamino
2
4
pyrazole solid product (3aꢀc) was obtained after recrystallization in EtOH (20 mL).
General Procedure B. Synthesis of Pyrazolodihydropyridine and Pyrazolopyridine derivatives.
To a solution of 1ꢀarylꢀ3ꢀindolylꢀ5ꢀamino pyrazole 3 (1.0 equiv.) in acetic acid (20 mL) was
added cyclic 1,3ꢀdiketone 4 (1.0 equiv.) and aryl aldehyde 5 (1.0 equiv.). The reaction mixture
was stirred at 100ꢀ110 °C for 2 h. After completion of the reaction (monitored by TLC), the
reaction mixture was cooled to room temperature. It was diluted with water (15 mL) and
neutralized with a saturated NaHCO solution (3 x 20 mL), extracted with EtOAc (3 x 20 mL),
3
dried over anhydrous sodium sulfate and concentrated under reduced pressure. The pure product
Pyrazolodihydropyridine or pyrazolopyridine (6 or 7) was obtained after recrystallization in
EtOH (20 mL).
General Procedure C. DDQ oxidation of Pyrazolodihydropyridines. To a solution of
pyrazolodihydropyridine derivative 6 (1.0 equiv.) in CH CN (20 mL) was added DDQ (1.0
3
equiv.). Then, the reaction mixture was stirred at room temperature for 2ꢀ3 h. After completion
of the reaction (monitored by TLC), the reaction mixture was diluted with water, extracted with
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dichloromethane (3 x 20 mL), dried over anhydrous sodium sulfate and concentrated under
reduced pressure. The crude product was purified by silica gel (60ꢀ120 mesh) column
chromatography by using hexane: EtOAc (80: 20) as an eluent.
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1
ꢀ(4ꢀChlorophenyl)ꢀ3ꢀ(1Hꢀindolꢀ3ꢀyl)ꢀ1Hꢀpyrazolꢀ5ꢀamine (3a). The title compound was
prepared according to General Procedure A using 3ꢀ(cyanoacetyl)indole 2 (300 mg, 1.63 mmol),
4
ꢀchlorophenyl hydrazine hydrochloride (321 mg, 1.79 mmol) and pꢀTsOH.H O (91 mg, 0.48
2
1
mmol) in EtOH to give 3a (401 mg, 80%); white solid; mp > 270 °C; H NMR (300 MHz,
DMSOꢀd ): δ = 11.22 (s, 1H), 8.18 (d, 1H, J = 7.2 Hz), 7.78 (d, 2H, J = 8.7 Hz), 7.71 (s, 1H),
6
7
.56 (d, 2H, J = 9 Hz), 7.40 (d, 1H, J = 7.5 Hz), 7.15ꢀ7.04 (m, 2H), 5.86 (s, 1H), 5.45 (s, 2H)
13
ppm; C NMR (50 MHz, DMSOꢀd ): δ = 148.0, 147.5, 138.6, 136.4, 129.4, 128.9, 124.9, 123.7,
6
ꢀ1
123.5, 121.4, 121.2, 119.3, 111.5, 109.4, 87.9 ppm; FTꢀIR (KBr, νmax/cm ) 3425, 1620, 1384,
+
1
069, 669; HRMS (ESI): m/z calcd for C H ClN [M + H] 309.0907; found 309.0902.
1
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1,4ꢀBis(4ꢀchlorophenyl)ꢀ3ꢀ(1Hꢀindolꢀ3ꢀyl)ꢀ7,7ꢀdimethylꢀ6,7,8,9ꢀtetrahydroꢀ1Hꢀpyrazolo[3,4ꢀ
b]quinolinꢀ5(4H)ꢀone (6a). The title compound was prepared according to General Procedure B
using 1ꢀ(4ꢀchlorophenyl)ꢀ3ꢀ(1Hꢀindolꢀ3ꢀyl)ꢀ1Hꢀpyrazolꢀ5ꢀamine 3a (300 mg, 0.97 mmol),
dimedone (136 mg, 0.97 mmol) and 4ꢀchloro benzaldehyde (136 mg, 0.97 mmol) in acetic acid
1
to yield 6a (430 mg, 80%); light yellow solid; mp >230 °C; H NMR (400 MHz, DMSOꢀd ): δ =
6
1
1.19 (s, 1H), 9.58 (s, 1H), 8.21 (d, 1H, J = 7.9 Hz), 7.75 (d, 2H, J = 8.8 Hz), 7.66 (d, 2H, J =
8.7 Hz), 7.43 (d, 1H, J = 2.6 Hz), 7.36ꢀ7.33 (m, 3H), 7.22 (d, 2H, J = 8.4 Hz), 7.12ꢀ7.08 (m,
H), 7.03 (t, 1H, J = 7.8 Hz), 5.37 (s, 1H), 2.56ꢀ2.44 (m, 2H, overlapped with DMSO residual
signal proved with HSQC), 2.20 (d, 1H, J = 16.1 Hz), 2.02 (d, 1H, J = 16.1 Hz), 1.0 (s, 3H),
1
13
0.82 (s, 3H) ppm; C NMR (75 MHz, DMSOꢀd ): δ = 193.9, 150.7, 145.4, 145.3, 137.5, 137.1,
6
135.8, 131.1, 130.1, 129.9, 129.3, 127.6, 125.1, 125.0, 124.3, 121.7, 119.4, 111.4, 109.9, 107.7,
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